7-{8 {60 -(2-aminomethyl-1,4-cyclohexadienyl)acetamido{9 -3-heterocyclic-thiomethyl-3-cephem-4-carboxylic acids and salts

ABSTRACT

7-( Alpha -(2-aminomethyl-1,4-cyclohexadienyl)-acetamido)-3heterocyclic-thiomethyl-3 -cephem-4-carboxylic acids, and their nontoxic, pharmaceutically acceptable salts and their Schiff bases, as made by reaction of salicylaldehyde with the free amino group, are valuable as antibacterial agents and are particularly valuable as therapeutic agents in poultry and animals, including man, in the treatment of infectious diseases caused by many Grampositive and Gram-negative bacteria.

United States Patent Naito et al.

[ Sept. 23, 1975 7-[a-(2-AMlNOMETHYL-l,4- CYCLOHEXADIENYL)ACETAMIDO1-3- HETEROCYCLlC-THlOMETHYL-3- CEPHEM-4-CARBOXYLIC ACIDS AND SALTS Inventors: Takayuki Nait'o, Tokyo; Jun

Okumura, Yokohama; Hideaki Hoshi, Tokyo, all of Japan Assignee: Bristol-Myers Company, New York,

Filed: Sept. 4, 1973 Appl. No.: 394,367

US. Cl 260/243 C; 424/246 Int. Cl C07d 99/24 Field of Search 260/243 C References Cited UNITED STATES PATENTS 10/1973 Lemieux et al. 260/243 C Primary ExaminerNicholas S. Rizzo Attorney, Agent, or Firm-Herbert W. Taylor, Jr.

[57] ABSTRACT mals, including man, in the treatment of infectious diseases caused by many Gram-positive and Gramnegative bacteria.

51 Claims, N0 Drawings 7-[a-(2-AMINOMETIIYL-1A- CYCLOIIEXADIENYL)ACETAMIDO]-3- I-IETEROCYCLIC-TIIIOMETI-IYL-3-CEPHEM-4- CARBOXYLIC ACIDS AND SALTS BACKGROUND OF THE INVENTION 1. Field of the Invention The cephalosporins of the present invention possess the usual attributes of such compounds and are particularly useful in the treatment of bacterial infections by parenteral administration.

2. Description of the Prior Art The cephalosporins are a well-known group of semisynthetic antibacterial agents made originally, for example, by acylation of the 7-amino group of the nucleus 7-aminocephalosporanic acid (7-ACA) and later by similar acylation of nuclei derived therefrom, as by modification of its substituent at the 3-position. Various reviews have appeared in the scientific literature (e.g. Cephalosporins and Penicillins-Chemistry and Biology, edited by Edwin H. Flynn, Academic Press, New York, 1972, and particularly pages 554-569) and in the patent literature, e.g. as in U.S. Pat. Nos. 3,687,948 and 3,741,965 (both U.S. Class 260-243C).

Recently issued patents on 3-thiolated cephalosporins in which the 7-substituent is a. a-Amino-a-phenylacetamido include U.S. Pat. No. 3,641,021, U.S. Pat. No. 3,734,907, US. Pat. No. 3,687,948, U.S. Pat. No. 3,741,965, Japan 7124400 (Farmdoc 46374S), Belgium 776,222 (Farmdoc 389831), Belgium 772,592 (Farmdoc 19696T), West Germany 2,202,274 (Farmdoc 50428T) corresponding to U.S. Ser. No. 167,534 filed Jul. 29, 1971, Netherlands 7205644 (Farmdoc 76309T); and

b. mor p-aminoethoxyphenylacetamido as Netherlands 72/13968 (Farmdoc 24740U) corresponding to U.S. Ser. No. 189,369 filed Oct. 14, 1971; and

c. o-aminomethylphenylacetamido as Netherlands 72/06326 (Farmdoc 76374T corresponding to U.S.

Pat. Nos. 3,766,175 and 3,766,176 issued Oct. 16, 1973) (which also reviews the older patent literature concerning substituted 7-phenylacetamidocephalospodoc 46,374T), Belgium Pat. No. 776,222 (Farmdoc 38,983T).

Recently issued patents containing a partially hydrogenated benzene ring in the 7-substituent but lacking a thiomethyl group at the 3-position include those in which the 7-substituent is a. 2-(1,4-cyclohexadiene-l-yl)acetamido as in U.S. Pat. No. 3,704,297 (Farmdoc 78154T) and Belgium Pat. No. 759,326 (Farmdoc 38,172S); and

b. a-amino-a-(l,4-cyclohexadienyl)acetamido and related compounds as in U.S. Pat. No. 3,485,819; West Germany 2,152,745 (Farmdoc 29606T); and

c. oz-amino-a-( 1,2-cyclohexenyl)acetamido as in Belgium 773,773 (Farmdoc 25515 T); and

d. p-(a-aminoalkyl)phenylacetamido as in U.S. Pat. No. 3,382,241; and

e. o-aminomethylphenylthioacetamido as in U.S. Pat. No. 3,657,232.

SUMMARY OF THE INVENTION This invention comprises the compounds of the formula CHZNH2 OOH wherein R is k iiLNllis s a 6H and their nontoxic, pharmaceutically acceptable salts and Schiff bases, as with salicylaldehyde.

In the preferred embodiment of the present invention I ll Ris N/N Such salts include carboxylic acid salts including nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts, e.g. salts of such nontoxic amines as trialkylamines, including triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, l-ephenamine, N,N'-dibenzylethylenediamine, dehydroabietylamine, I N,N'-bisdehydroabietylethylenediamine, N-(lower)- alkylpiperidine, e.g. Nethylpiperidine, and other amines which have been used to form salts with benzylpenicillin; and in all cases the nontoxic, acid addition salts thereof (i.e., the amine salts) including the mineral acid addition salts such as the hydrochloride, hy-

drobromide, hydroiodide, sulfate, sulfamate and phosvphate and the organic acid addition salts such as the maleate. acetate, citrate, oxalate, succinate, benzoate,

tartrate. fumarate, malate, mandelate, ascorbate and the like.

The compounds of the present invention are prepared according to the present invention by coupling with a compound of the formula H,N 5 e i N y CH2S-R o 1 u I COOH wherein R has the meaning set out above (or a salt or easily hydrolyzed ester thereof including those of US. Pat. No. 3,284,451 and U.K. Pat. No. 1,229,453 and any of the silyl esters described in US. Pat. No.

3,249,622 for use with 7-aminopenicillanic acid and I used inGreat Britain Pat. No. 1,073,530) a particular acid or its functional equivalent as an acylating agent for a primary amino group. After coupling, the blocking group is removed to give the desired product. Said acid has the formula cH,cooH

or a B-diketone as in Great Britain Pat. No. 1,123,333, e.g. methyl acetoacetate, in which case the acid containing the blocked amino group is preferably converted to a mixed anhydride, as with ethyl chloroformate, before reaction with compound II or a salt thereof to form the desired product 1 after acid cleavage. 1

Further to the discussion above of blocking groups used on the free amino group of the sidechain acid during its coupling with compound II, the blocking group is then removed to form the products of the present invention, e.g., the t-butoxy-carbonyl group is removed by treatment with formicacid, the carbobenzyloxy group is removed by catalytic hydrogenation, the 2-hydroxy-l-naphthc'arbonyl group is removed by acid hydrolysis and the trichloroethoxycarbonyl group by treatment with zinc dust in glacial acetic acid. Obvious ly, other functionally equivalent blocking groups for an amino group can be used and such groups are considered within the scope of this invention.

Thus. with respect to said acid to be used to couple ters of carbonicacid, or alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester of thioester (e.g., with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with compound 11 after first reacting said free acid with N,N-dimethylchlor oformiminium chloride [cf. Great Britain, 1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or by the use of enzymes or of an N,N'- carbonyldiimidazole or an N,N-carbony1ditriazole [cf. South African patent specification 63/2684] or a carbodiimide reagent [especially N,N-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N- cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; cf. Sheehan and Hess, J. Amer. Chem. Soc., 77, 1067 (1955)] or of alkylamine reagent [cf. R. Buijle and H. G. Viehe, Angew-Chem. International Edition 3, 582

(1964)], or ofa ketenimine reagent [cf. C. L. Stevens and MJE. Mond, J. Amer. Chem. Soc., '80, (4065)- or of an isoxaz'olium salt reagent [cf. R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc. 83, 1010 (1961 )1. Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of an quasiaromatic five-membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'- carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield dimidazolide. The by-product, imidazole, precipitates and may be separated and the imidazolide isolated, but this is not essential. The methods for carrying out these reactions to produce a cephalosporin and the methods used to isolate the cephalosporin so produced are well known in the art.

3 In the treatment of bacterial infections in man, the compounds of this invention are administered parenterally, in accordance with conventional procedures for antibiotic administration, in an amount of from about 5 to 200 mg./kg./day and preferably about 5 to 20 mg./kg./day in divided dosage, e.g., three to four times a day. They are administered in dosage unit s containing, for example, or 250 or 500 mg. of active ingredientwith suitable physiologically acceptable carriers or excipients. The dosage units are in the form of liquid preparations such as solutions or suspensions.

Certain 3-substituted 7-[a-(2-aminometh ylphenyl)- acetamido]cepha1osporanic acid derivatives (A; see Netherlands 72/06326, Farmdoc 76374T, corresponding to US. Ser. No. 142,337 filed May 11, 1971 and isvery effective derivatives with a broad spectrum of activity. Their limited water solubility 2 mg./ml.) has,.

however, caused crystalluria formation evenwhen the antibiotics had been administered parenterally as a readily-dissociable soluble derivative. The object of the There is provided by the present invention certain 7- [a-( 2-aminomethyl- 1 ,4-cyclohexadienyl )acetamid01- cephalosporanic acids (B).

CH NH present invention was, therefore, to obtam equally ac- 5 tive compounds which show higher water solubility as the. zwitterion form.

H CH- CO- s C Z Z IO CH2SR 0 COQH H CH CON S 15 (B) N Many of the new series were found to be more solu- CH ble than the corresponding phenyl derivatives and to 0 have a solubility of more than 7 mg./ml. determined CO H routinely in 0.1 M pH 7.0 phosphate buffer, as shown 20 below compared with that of certain corresponding (A) phenyl derivatives.

CH NH z z CHICO-N s CH CO-N s CH2S-R CH S-R O 0 COOH COOH R Solubility* Solubility* N OH Ex. I 4.1 mg./ml. 4.4 mgJml. (I)

I'm {3 h Ex. 2 8.7 mg./ml. 3.5 mgJml. (2)

l vfl Ex. 3 9.7 mg./ml. 1.9 mg./ml. (3)

ls l 3 Ex. 4 7.6 mg /ml. 0.9 mgJml. 4

N Ex. 5 7.7 mg/ml. 3.6 mgJml. I N

I V (N I Ex. 6 9.0 mg./ml. 30 mgJml.

Continued CH NH CH NH S S CH CO-N CH,CON

CH,-S--R O OOH COOH R Solubility" Solubility 2 I Ex. 7 8.4 mgJml. 3.7 mgjml.

Stirred at 25 C. for 4 hours in (H M pH 7.0 phosphate buffer-filtered through Milipore filter (0.45 yJ-assayed spectroscopically against its own standard.

(I) This compound (also called BB-SISO) is claimed in U.S.S.N. 285,764 filed August 31. 1972 (2) This compound (also called Bil-S226) is claimed in U.S.S.N. 284.792 filed August 30. I972 (3) This compound (also called MR-S94) is claimed in U.S.S.N. l42.337 filed May ll. l97l (see Netherlands 72/063236; Farmdoc 76.374T). (4) This compound (also called MR-S96) is claimed in U.S.S.N. 142.337 filed May l l. l97l (sce Netherlands 72106326; Farmdoc 76.3741).

Preparation of 7-[a-(2-Aminomethyl-l,4-cyclohexadienyl)acetamidolcephalosporanic acid derivatives cum, CHZNH,

Li/Liq NH,

CH,COOH cmcoon t-BuCON; cuacocrucoom 2 CH3 CH,NH-t-BOC CH,NH =CHCO0Et cu coou u coou 2,4-DNP/DCC ClCOOEt CH,NH-t-BOC H,NHC=CHCOOEt cmcooigqio, cu,co o cooer s s HgN 2N H,SR CH,SR 0 o co ,H o,|-|

CH -N=CHb H s s HZCON CHZCON CH2-SR C I R i our 02H Continued Preparation of 7-[a-(2-Aminomethyl-1.4-cyclohexadienyl)acetamido]cephalosporanic acid derivatives a b c d Amberlite IR-120 and is a strong cation exchange resin containing sulfonic acid radicals. Amberlite 1R-120 is a commercially available cation exchange resin of the polystyrene sulfonic acid type; it is thus a nuclear sulfonated polystyrene resin cross-linked with divinyl benzene obtained by the procedure given by Kunin, [on Exchange Resins, 2nd. Edition (1958), John Wiley and Sons, Inc. Therein see pages 84 and 87 for example.

Preparation of Starting Materials a-(Z-Aminomethyl-l ,4-cyclohexadienyl)acetic acid A solution of 16.5 g. (0.1 mole) of o-aminomethyl- -phenylacetic acid in 1.5 lofliquid ammonia (which had been treated with 50 mg. of Li to remove a trace of moisture) was slowly diluted with 500 ml. of dry t- BuOH. To the solution was added in small portions 3.4 g. (0.5 atom) of Li over a period of 4 hours and the mixture was stirred for 16 hours at room temperature removing the liquid ammonia in a hood and finally evaporated to dryness below 40 C. The residue was dissolved in 500 ml. of water and the solution was chromatographed on a column of IR-l20 (H*, 700 ml.) resin and eluted with 1% NH OH solution. Ninhydrin positive fractions of the eluate were combined and evaporated to dryness. The residue was washed with four 50 ml. portions of hot acetone and recrystallized from 500 ml. of ethanol-water (1:1) to give 11.2 g. (67%) of colorless needles, 2. M.p. 183 C.

1R:v,,,,,,""1630, 1520, 1380, 1356 cm.

Found: C, 64.77; H, 8.06; N, 8.44.

Alternative Procedure for the Preparation of a-( 2-amin0methyl-1 ,4-cyclohexadienyl)-acetic acid Anal. Calcd. for C9H13NO2: c, 64.65; H, 7.84; N,

The procedure used by Welch, Dolfini and Giarrusso in US. Pat. No. 3,720,665 (Example 1) to make D-2- amino-2-(1,4-cyclohexadienyl)acetic acid was adapted. A solution of 830 ml. of distilled liquid ammonia was dried with 40 mg. of lithium under an argon atmosphere To this stirred solution was added 11.0 g.

.(0.07 mole) ofsZ-aminomethylphenylacetic acid and 340 ml. of tert. buty1 alcohol. A total 01 1.6 g.-(0.225 mole) of lithium was added to the vigorously stirred solution over a period of 2 hours. The grey mixture was then treated with 35 g. (0.215 mole) of triethylamine (TEA) hydrochloride and stirred overnight at room temperature for 18 hours. The tert. butyl alcohol was removed at 40 15 mm.) to yield a white residue which was dried in vacuo over P 0 ovenight. The solid was dissolved in 30 ml. of 1:1 methanol-water and added with stirring to 3.5 1. of 1:1 chloroform-acetone at 5. The mixture was stirred for 20 min. and theamino acid 2, d-(Z-aminomethyl-l,4-cyclohexadienyl)acetic acid, was collected and dried for 16 hours in vacuo over P 0 to yield 6.3 g. (58%) of white crystals, m.p. 190 decomp. The [R and NMR spectra were consistent for the structure.

a-[2-(t Butoxycarbonylaminomethyl)-1,4-cyclohexadienyl]acetic acid (3) To a stirred solution of 8.0 g. (0.048 mole) of 2 and 3.8 g. (0.096 mole) of NaOH in 150 ml. of water was added a solution of 10.3 g. (0.072 mole) of tbutoxycarbonylazide in ml. of THF and the mixture was stirred for 18 hours at room temperature. The THF was removed under reduced pressure and the residual solution was washed with ether (2 X ml.), acidified with 6 N HCl and extracted with ether (3 X 100 ml The combined extracts were washed with water (2 X 100 ml.) and a saturated NaCl solution (100 ml.), dried with Na SO and evaporated to dryness. The oily residue was triturated with n-hexane to give 10.5 g. (82%) of colorless powder 3 melting at 1 13 C.

IR: v 3370, 1715, 1640, 1530, 1280, 1160 cm".

NMR: 8,,,,,,,""a 1.45 (9H, s, t-Bufl), 2.73 (4H, s, H C 3.16 (2H, s, C1;1 CO), 3.76 (2H. d, 61-12, Cfl N) 4.90 (1H, m, Nlj), 5.66 (2H, s, C=), 10.6 (1H, br-s, COOL-1).

Anal. Calcd. for C H NO C, 62.90; H, 7.92; N, 5.24 i Eound c, 63.13; H, 8.21; N, 5.26.

Sodium{2-[N-( l-carbethoxypropen-2- yl)aminomethyl]-1,4-cyclohexadienyl}acetate (4) To a stirred solution of 460 mg. (0.02 mole) of metalhe sodium in 100 ml. of absolute EtOH was added 3.34 g. (0.02 mole) of2 and 3.1 g. (0.024 mole) of ethyl acetoacetateand the mixture was heated to reflux for 4 hours with stirring. The hot reaction mixture was filtered and the filtrate was allowed to keep cold overnight to give 2.0 g. of colorless needles 4 melting at 264 C. The additional product (3.3 g.) was obtained by concentration of the mother liquid. The total yield was 5.3 g. (88%).

IR: v 3300, 1635, 1600, 1570, 1300, 1275, 1170, 1090 cm.

NMR: 8 ,."z" 1.23 (3H, t, 7H2, CH CH 1.96 and 2.25 (3H, s, C='C-CIi cis and trans), 2.70 (4H, s, lj C 3.04 (2H, s, CH CO), 3.66 and 3.95 (2H, s, CH -N, cis and trans), 4.07 (2H, q, 7H2, Cl;l CH 4.45 and 4.56 (l=s, 32 cis and trans), 5.76 (2H, s,

Anal. Calcd. for C H NO Na: C, 59.79; H, 6.69; N, 4.64

Found: C, 59.69; H, 6.76; N, 4.75.

7-Amino-3-(3-hydroxypyridazin-6-ylthiomethyl)-3- cephem-4-carboxylic acid (5a) HQ- l l O N CH2-S OH I 60 1-1 3 -Chloro-6-hyd roxypyridazine A mixture of 22.47 g. (0.15 mole) of 3,6- dichloropyridazine and 50 ml. of acetic acid was refluxed for two hours. The reaction mixture was cooled and diluted with 50 ml. of water and then concentrated to dryness under reduced pressure. The residue was crystallized from water to give 15.8 g. (80%) of 3-chloro-6-hydroxypyridazine as colorless prisms which melted at l337 C. (lit. 138l40 C.). See N. Takabayashi, Yakugaku Zasshi, 75, 778 (1955).

3-Hydroxy-6-mercaptopyridazine A mixture of 2.6 g. (0.02 mole) of 3-chloro-6- hydroxypyridazine and 5.0 g. (0.07 mole) of freshly prepared potassium hydrogen sulfide in ml. of ethanol was heated atl30-l40 C. in a sealed tube for 6 hours. The reaction mixture was cooled and diluted with 200ml. of water. Almost all organic solvent was removed by distillation under reduced pressure. The residual aqueous solution was acidified with dilute hydrochloric acid to pH 3 and extracted with ethyl acetate (6 X 50 ml.). The combined extracts were evaporated to dryness and the residue was reprecipitated from 30 ml. of ethanol-ligroin (1:1) to give 2.2 g. (87%) of amorphous 3-hydroxy-6-mercaptopyridazine. m.p. 158 159 C. (lit. 157-158 C.).

See .1. Druey et al., Helv. Chem. Acta. 37, 121 (1954).

3-Hydroxy-6-mercaptopyridazine To a solution of 5.6 g. (0.05 mole) of 3,6- dihydroxypyridazine in 150 ml. of pyridine was added portion-wise 2.70 g. (0.012 mole) of phosphorus pentasulfide with vigorous stirring under refluxing. The refluxing was continued for one hour and then the reaction mixture was diluted with 200 ml. of water and concentrated to remove the pyridine. The resulting oily residue was suspended in water and extracted with ethyl acetate. The organic extracts were combined and concentrated again to give oily material which was triturated with a small amount of water to give 3-hydroxy- 6-mercaptopyridazine as a yellow solid. Recrystalli-- zation from water afforded 0.62 g. (12%) of the prod uct which was identical with that prepared above.

3 ,6-Dihydroxypyridazine To a boiling solution of 315 g. (3 moles) of hydrazine dihydrochloride in 2 L of water was added portionwise 295 g. (3 moles) of finely ground maleic anhydride with stirring. After the addition was completed the heating was continued for 4 hours and then allowed to stand overnight in a refrigerator to give 285 g. of 3,6-dihydroxypyridazine as massive pillars. m.p. 290 C.

3 ,6-Dichloropyridazine A mixture of g. (1.33 moles) of 3,6- dihydroxypyridazine and 250 g. of phosphorus oxychloride wasrefluxed for 3 hours under protection from moisture. The excess of phosphorus oxychloride was removed under reduced pressure and the dark residue was poured into one Kg. of crushed ice. The resulting precipitate was collected by filtration. The second crop of the product was obtained from the mother liquor by extraction with five 300 ml. portions of chloroform followed by treating with 1 g. of charcoal and evaporating the solvent. The first and second crops were combined, dissolved in 500 ml. of chloroform and treated again with one g. of charcoal and concentrated to give g. (83%) of 3,6-dichloropyridazine as fine needles melting at 606l C. (in a sealed tube).

6-Chloro-3-hydroxypyridazine A suspension of 60 g. (0.4 mole) of 3,6- dichloropyridazine in 200ml. of 10% hydrochloric acid was refluxed for 2 hours until a clear solution was obtained. The clear solution was treated with ca. 1.5 g. of active carbon and filtered. The filtrate was concentrated under reduced pressure to give 6-chloro-3- hydroxypyridazine as colorless needles. Yield 49.5 g. (98%). M.p. l37139 C.

3-Hydroxy-6-mercaptopyridazine A mixture of 50 g. (0.38 mole) of 6-chloro-3- hydroxypyridazine, 60 g. (0.83 mole) of potassium hydrogen sulfide in 250 ml. of ethanol was heated in a 500 ml. autoclave at 140 C. for 14 hours with magnetic stirring. The pressure reached to l520 Kg./cm The mixture was evaporated to dryness and the residue was dissolved in 300 ml. of water. The aqueous solution was acidified with dil hydrochloric acid to pH 3 and extracted with ten 200 ml. portions of ethyl acetate. The combined extracts were concentrated to give 34.3 g. (70%) of amorphous 3-hydroxy-6-mercaptopyridazine.

7-Amino-3-(6-hydroxypyridazin-3-ylthiomethyl)-3- cephem-4-carboxylic acid A mixture of 0.60 g. (0.0047 mole) of 3-hydroxy-6- mercaptopyridazine, 1.27 g. (0.0047 mole) of 7-aminocephalosporanic acid, 0.78 g. (0.0094 mole) of buffer (pH 6.4) was heated at 60 C. for hours. The reaction mixture was filtered to remove a trace of insoluble material and the filtrate was adjusted to pH 5 with acetic acid to give brown precipitates, which were collected by filtration, washed with water and acetone successively and dried in vacuo to give 7-amino-3-(6- hydroxypyridazin-3-ylthiomethyl)-3-cephem-4- carboxylic acid; 1.03 g. (71%). M.p. 290-300 C. (decomp.).

lRz v 1805, 1680, 1650, 1580, 1415 cm UV: A 249 nm (519400) NMR: 8,,,,,,,"z""a"'s 3.22 (1H, d, 19 Hz), 3.37 (1H, d, 14 Hz), 3.65 (1H, d, 14 Hz), 3.72 (1H, d, 19 Hz), 4.90.( 1H, d, 4 Hz), 5.30 (1H, d, 4 Hz), 6.75 (1H, d, Hz), 7.30 (1H, d, 10 Hz).

Anal. Calcd. for C H N O S J/2H O: C, 41.25; H, 3.75; N, 16.04; S, 18.36

Found: C, 41.45; H, 3.70; N, 15.83; S, 18.03.

7-Amino-3-( 3-hydroxypyridazine-6-ylthiomethyl )-3- cephem-4-carboxylic acid A mixture of 141 g. (0.52 moles) of 7-ACA. 92 g. (1.1 moles) of sodium bicarbonate and 73 g. (0.57

moles) of 3-hydroxy-6-mercaptopyridazine in 1.5 L of 7-Amino-3-(tetrazolo[1,S-b]pyridazin-6-ylthiomethyl- 3-cephem-4-carboxylic acid (5b) a :N :H2N I o GHQ-SUN 00 a Preparation of 6-Mercaptotetrazolo[4,5-b]pyridazine POCl 1 n O 5 .9.

NH NH ale. KSH

14 3,6-Dihydroxypyridazine (9) To a boiling solution of 315 g. (3 moles) of hydrazine dihydrochloride in 2 L of water was added portionwise 295 g. (3 moles) of finely ground maleic anhydride 8 with stirring. After the addition was completed the heating was continued for 4 hours and then allowed to stand overnight in a refrigerator to give 285 g. (85%) of 9 as massive pillars. m.p. 290 C.

3.6-Dichloropyridazine 10) A mixture of 150 g. (1.33 moles) of 9 and 250 g. of phosphorus oxychloride was refluxed for 3 hours under protection from moisture. The excess of phosphorus oxychloride was removed under reduced pressure and the dark residue was poured into one Kg. of crushed ice. The resulting precipitate was collected by filtration. The second crop of the product was obtained from the mother liquor by the extraction with five 300 ml. portions of chloroform followed by treating with 1 g. of charcoal and evaporating the solvent. The first and second crops were combined, dissolved in 500 ml. of chloroform and treated again with one g. of charcoal and concentrated to give 165 g. (83%) of 10 as fine needles melting at -61 C. (in a sealed tube).

3-Chloro-6-hydrazinopyridazine (1 l A mixture of 40 g. (0.27 mole) of 3,6- dichloropyridazine (10) and 40 ml. of hydrazine hydrate in 80 ml. of ethanol was refluxed for 1 hour. The reaction mixture was evaporated to dryness and the residue was recrystallized from benzene to give 39 g. of 11 melting at 1l4-115 C.

6-Chlorotetrazolo[4,5 -b]pyridazine 12) To a solution of 25.7 g. (0.174 mole) of 11 in 100 ml. of 15% acetic acid was added dropwise a solution of 13.8 g. (0.2 mole) of sodium nitrite in 50 ml. of water with vigorous stirring at 5-10 C. Stirring was continued for 1 hour at the same temperature. The precipitate which separated was filtered, washed with 20 ml. of water and air-dried to give 17.02 g. of 12. Additional product was obtained by evaporation of the filtrate. Total yie1d 18.32 g. (64%). M.'p. 104105 C.

6-Mercaptotetrazolo[4,5-b]pyridazine 13) A mixture of 21.3 g. (0.137 mole) of 12 and 20g. (0.25 mole) of potassium hydrosulfide in 200 ml. of

N- -N MUM gig ethanol was refluxed for 2 hours and evaporated to dryness. The residue was dissolved in 100 ml. of waterand filtered to remove a small amount of insoluble material. The filtrate was acidified to pH 1 with'dil. hydrochloric nAnal. Calcd. forC acid to precipitate 13 as colorless needleswhich were 5 26.83; S. 17.5.5

collected by filtration, washed with 20 ml. of water and dried. Yield 9.80 g. (47%). M.p. 140141 C. (dec.). 1R: 2500, 1540, 1445, 1295, s40 m .f NMR: m 7.44 (1H, d, 10 Hz, pyridazine- E), 7.77 (1H, d, '10 Hz, pyridazine-fl). Anal. Calcd. for C H N S: C, 31.37; H, 1.97; N, 45.72; S, 20.94 v Found: C, 3l.52';'31.66; H, 1.70, 1.69; N, 46.01; 46.01; S, 20.95.

- Preparation of 7-amino-3-(tetrazolo[4,5-b]pyridazin-6-ylthiomethyl)- 3-cephem-4-carboxylic acid HgN s 1 I H ta o-coca, H

7-ACA 1 solve insoluble material. The solution was treated with active carbon, filtered and the filtrate was acidified to pH 5 with dil. hydrochloric acid. The precipitate was collected by filtration, washed with water, air-dired and finally in vacuo on P 0 to give 14.47 g. (64%) of 5b. M.p. 248-250 C. (dec.).

(ii) A stirred solution of 16.8 g. (0.11 mole) of 13 and 18.48 g. (0.22 mole) of NaHCO in l L of 0.1 M

phosphate buffer (pH 6.4) was heated at 50 C. and to the solution was added portionwise 30 g. (0.11 mole) of 7-ACA. The mixture-was heated at 80 C. for 2.5 hours, during which period insoluble material still re mained. The reaction mixture was cooled to room tem-; perature and the precipitated 5b was collected by filtration, washed thoroughly with 200 ml. of water and air-, dried.

Additional 5b was obtained from the filtrate and the washings by acidifying to pH 5 with dil. HCl. Total yield 32.9 g. (83%). M.p. 245-250 C. (dec.).

IR: v,,,,,, 1800, 1615, 1538, 1360 cm* UV: k a 237 nm (6 19500), 275 nm (e 12000), 310 nm (sh) (e 5700).

NMR: 8,,,,,,,"2"*"2""a 3.35 (1H, d, 18 Hz, 2-H), 3.76 (1H, d, 18 Hz, 2-l;l), 4.00 (1H, d, 10 Hz, 3-CL 2). 4.48

.Found: c, 39.19;-H, 271,11, 26.84; s. 17.35. 1

7. Amino-3-(5-inethyl -1,3,4-thiadiazol-2 Y g ylthiomethyl)-3-cephem 4-carboxylic acid (5d) Z-Mercapto-S-methyll ,3,4-thiaziazole Lit. ref. U.S. Pat. No. 3,516,997 (1970); Antibiotics, 23, 131-36,(1970). 4

11.5 g. (0.1 mole of 2-amino-5-methyl-l,3,4- thiadiazole was carefully ground together with '32 g. (0.45 mole) of sodium nitrite and slowly added to 160" ml. of-48% HBr containing 50 mg. of powdered copper at -l0 C. with stirring. After the addition was completed, the solution was stirred at 5 C. for 1 hour and then at 20C. for 1V2 hours. ThepH was adjusted to 9:5 by addition of 50% KOH and the solution was heated to C. At 60 C. the pH was readjusted to 9.5 by ad .dition of'50% 'KOH. The-solution was cooled and filteredJTheprecipitate was dissolved inether and the filtrate'was extracted with 2x 200 ml. et'heri'Fhe cor'hbined ether solutions were dried over sodium sulfate and evaporated to dryness. The product was recrystallized from benzene-Skellysolve 13.1 Yield 12 g,, m.p. 105"107'C. 4 I I w 1 I 12 g. .07 mole) 6f 2-bromo-5-methyl-1,3,4- thiaziazoland 5 g. (.07 mole) of thiourea were dissolved in 40 ml. of 100% ethanol and refluxed for one and a half hours 011 a steam bath. This solution as added to 4.5 g5 (.08 mole) of KOH in ml. H O and the mixture heated tcg boiling for five minutes. The ethanol was removed under vacuum and the pH of the aqueous solution adjusted to 3 by addition of 3 N HCl. The product crystallized out and after cooling at, 0 C. for 1 hour was collected by filtration, washed with cold water and recrystallized from ethanol. Yield 5 g., "m.p. 186187 c. 1

Anal..-Calcd. for C H N S C, 27.25; H, 3.05;;N, 21.19; 5, 48.51 Found: C, 27.20; H, 3.34; N, 21.18; S, 4848' 7-Amino-3-(5-methyl-l,3,4-thiadiazol-2- ylthiomethyl)-3-cephem-4-carboxylic acid. 1

To a stirred suspension of 2.72 g. (0.01 mole) of in 50 ml. of 0.1 M, pH 6.4 phosphate buffer,

was added 1.68 (0.02 mole) of NaHCO followed by 1.45 g. (0.01 1 mole) of 2-mercapto-5-methyl-l ,3,4- thiadiazole and the mixture heated and stirred at 60 C. for 5 hours. The resulting slurry was then allowed to cool to about 22C. over a 1 hour period. The crystalline precipitate was collected by filtration, washed with water and air dried. Yield 1.3 g., dec. pt. 206 C. Seal ing up the reaction 10X: ga'v'e 18.0 g.

Anal. Calcd. for C' H, N O S C. 38.37; H, 3,52; S, 27.96.

Found: C, 39.06; H, 3.91; S, 26.67.

7-Amino-3-( 1-methyltetrazol'5-ylthiomethyl)-3- cephem-4-carboxylic acid (5c) T'l N cu sk o 02H N 1 CH,

The above preparation was repeated using l-methyll,2,3,4-tetrazole-5-thiol instead of the thiadiazole. There was obtained 25 g. (76%) of 7-amino-3-(lmethyl-1,2,3,4-tetrazole-5-thiomethyl)-A -cephem-4- carboxylic acid. The preparation of this same compound is also described in US. Pat. No. 3,516,997 in column 6 under the heading Preparation 7.

7-Amino-3-( 3-hydroxypyridazino[ 3 ,2-c]s-triazol-6- ylthiomethyl)-3-cephen-4-carboxy1ic acid (5e).

HEN H l O/ N ca -s- =N 6-Mercapto-2,3-dihydro-s-triazolo[ 4,3-bj-pyridazin- 3-one A mixture of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]- pyridazin-3-one [P. Francavilla and F. Lauria, J. Het.- Chem., 8, 415 (1971) (1.70 g., 0.01 mole) and potassium hydrosulfide (1.44 g., 0.02 mole) in ethanol (30 ml.) was heated in a sealed tube for 8 hours at 140. After cooling, water (50 ml.) was added to the reaction mixture and a small amount of insoluble material was removed by filtration. The filtrate being concentrated under reduced pressure, the concentrate was acidified to pH 2 with dil. HCl to afford yellow precipitate of 6- 7-Amino-3-[2,3-dihydro-s-triazolo[4,3-b]pyridazin-3- one-6-ylthiomethyl]-3-cephem-4-carboxylic acid (e) 7-ACA (1.36 g., 5 mmoles) was added at 50 C. to a solution of 6-mercapto-2,3-dihydro-s-triazolo[4,3- b]pyridazin-3-one (0.84 g., 5 mmoles) and sodium bicarbonate (0.84 g., 10 mmoles) in ml. of 0.1 M phosphate buffer solution (pH 6.4) and the mixture was heated for 2 hours at 70 C. A small amount of insoluble material was removed by filtration and acidification of the filtrate to pH 5 with dil. HCl afforded the product 5e which was collected by filtration, washed with water (30 ml.) and dried in vacuo on P 0 Yield of 7-amino-3[2,3-dihydro-3-triazolo(4,3-b)pyridazin- 3-one-6-ylthiomethyl]-3-cephem-4-carboxylic acid 1.25 g. (66%), M.p. 300 C.

IR: v,,,,,,"' 1805, 1720, 1620, 1550 cm UV: A H 257 nm (617700) NMR: 8,,,,,,,2""2""s 3.40 (1 H, d, 20 Hz, 2-lj), 3.78 (1 H, d, 20 Hz, 2-H), 4.00 (1 H, d, 13 Hz, 3-CH 4.35 (1 H, d, 13 Hz, 3-Cfl 5.02 (1 H, d, 4 Hz, 6-H), 5.40 (1 H, d, 4 Hz, 7- l;l), 6.70 (1 H, d, 9 Hz, pyridazine-lj), 7.40 (1 H, d, 9 Hz, pyridazine-H).

Anal. Calcd. for C H N O S H O: C, 39.19; H, 3.54; N, 21.09; S, 16.09

Found: C, 39.40; H, 3.39; N, 20.36; S, 15.89.

7-Amino-3-(pyrido[2,l-c]s-triazol-3-ylthiomethyl)-3- cephem-4-carboxylic acid (5f) H N -N i it ig ca sg I 0 H To a hot (60) solution of 3-mercapto-s-triazolo[4,3- a]pyridine [D. S. Tarhell et al., J. Am. Chem. Soc. 70, 1381 (1948)] (1.51 g., 10 mmole) and NaHCO (1.68 g., 20 mmole) in 0.1 M pH 7.4 phosphate buffer (50 ml.) was added portionwise 7-ACA (2.72 g., 10 mmole) and the mixture was heated at -85 for 30 min. The reaction mixture being treated with carbon, the filtrate was acidified to pH 5 with dil. HCl to give 7-amino-3-(s-triazolo[4,3-a]pyridin-3-ylthiomethyl)- 3-cephem-4-carboxylic acid which was collected by filtration, washed with water (10 ml.) and dried in vacuo on P 0 Yield 1.40 g. (39%). M.p. 215-220 (dec.).

1R: v 1805, 1620, 1530, 1410, 1545 cm "(1H, d, 13 HZ, 3-lj), 3.68 (1 H, d, 18 Hz, 2-H), 4.18

(1 H, d, 13 Hz, 3-11), 4.7-5.3 (2 H, m, 6-l;1 and 7-H). Anal. Calcd. for C H N O S C, 46.27; H, 3.61; N, 19.27; S, 17.65

Found: C, 45.81; 45.74; H, 3.58, 3.69; N, 18.21, 18.13; S, 17.08.

7-Amino-3-pyridazino[2,1-c]-s-triazol-3- ylthiomethyl)-3-cephem-4-carboxy1ic acid (5g) -N g ca si 1 3-Mercapto-s-triazolo[4,3-b]pyridazine Anal. Calcd. for C H N S.1/2H O: C, 37.26; H, 3.13; N, 34.76

Found: C, 37.35; H, 2.32; N, 34.81.

7-Amino-3-( s-triazolo[4,3-b]pyridazin-3- ylthiomethyl)-3-cephem-4-carboxylic acid (5g) 7-ACA 1.36 g., 5 mmole) was added portionwise to a solution of 0.68 g. (4.5 mmole) of 3-mercapto-striazolo-[4,3-b]pyridazine and 0.84 g. (10 mmole) of NaHCO- in ml. of 0.1 M pH 7.4 phosphate buffer 20 at 4050. The mixture was heated at 80-85 for 40 min., treated with a small amount of active carbon and acidified with dil. HCl to pH 4 to precipitate 7-amino-- 3-(s-triazolo[4,3-b]-pyridazin-3-ylthiomethyl)-3- cephem-4-carboxylic acid which was collected, washed 2 with 20 ml. of water and dried in vacuo on P 0 to yield 0.81 g. (49%), M.p. 300".

IR: v 1705, 1620, 1540, 1415, 1350 cm".

UV: A,,,,,, ""a 275 nm (e13100) NMR:cr,,,,,,,"1" -z""a 3.35 1 11,11, 18 Hz, 2-1 1), 3.86 (1 H, d, 18 Hz, 2-l;1), 4.80 (l H, d-d, 4 and 2 Hz, 6-lj), 5.24 (l H, d, 4 Hz, 7-l l), 7.1-8.6 (3 H, m, pyridazine- Anal. Calcd. for C H N O S .H O: C, 40.83; H, 3.69; N, 21.96; S, 1678 Found: C, 41.15; H, 3.24; N, 20.37; S, 17.90.

7-Amino-3-( ,2,3-triazol-5-ylthiomethyl)-3-cephem-4- carboxylic acid (5h) COOH Synthesis of potassium 1,2,3triazole-5-thiolate The synthesis of the thiol was accomplished by a procedure essentially identical to that described in the literature [.l. Goerdler and G. Gnad, Chem. Ber. 99, 1618 5-Benzamido-1 ,2,3-thiadiazole To a stirred solution of benzoylisothiocyanate (50.6

g., 310 mmoles) in commercial anhydrous ether (400 ml.), maintained at 0 and in a nitrogen atmosphere, was added dropwise with vigorous stirring, 0.685 N ethereal diazomethane (453 ml., 310 mmoles). When the addition was completed, the mixture was stirred for 1 hour at 0, the solid was collected by filtration and dried in vacuo. The melting point of the crude material (23.3 g.) thus obtained was observed somewhere in the region 232 to 257. Goerdler reported M.p. 267 for the pure material. A' small second crop (2.1 g.) was obtained by evaporation of the mother liquor in vacuo. The total yield was therefore 40%.

1,2,3-Tr1az616-5-1h161 A solution of the above benzamido compound (8.2 g., 40 mmoles) in 2 N sodium hydroxide ml., mmoles) was heated under reflux temperature in a nitrogen atmosphere for 24 hours. The solution was cooled to 0 in ice, and concentrated hydrochloric acid (26 ml.) was added, while a continous stream of nitrogen was passed through the solution. The benzoic acid which precipitated was collected by filtration; the filtrate was saturated with sodium chloride and the additional benzoic acid which separated was removed by filtration. The filtrate was immediately extracted with ethyl acetate, the extract was washed with saturated salt solution, dried over magnesium sulfate and then evaporated in vacuo. The viscous oil which remained was immediately evaporatively distilled in vacuo (7075/0.00l mm.) to give an oil (2.84 g., 70%) which solidified (m.p. 52-59; Goerdler reported m.p. 60) spontaneously.

Potassium 1,2,3-Triazole-5-thiolate To a solution of the above thiol (2.84 g., 28.1 mmoles) in absolute ethanol (28 ml.) was added 1.93 N alcoholic potassium hydroxide solution-(14.5 ml.). The solution was then diluted with anhydrous ether until crystallization of the salt was completed. The solid was collected by filtration, washed with ether, and dried in vacuo. The salt obtained in this manner (3.65 g., 93%) had m.p. 225 with decomposition.

7-Amino-3-[S-( 1 ,2,3-triazole-5 -yl)-thiomethyl]-3- cephem-4-carboxylic acid (5h) Ten grams (0.075 mole) of S-mercapto-l ,2,3triazole potassium salt was added to a stirred slurry of 19 g.

(0.07 mole) of purified 7-aminocephalosporanic acid and 5.9 g. (0.07 mole) of NaHCO in 350 ml. of 0.1M phosphate buffer (pH 6.4) and the mixture heated and stirred at 55 C. for 3 V2 hours under a nitrogen atmosphere. The resulting solution was cooled to 22 C(and the pH adjusted to 5.5 with 40% H PO The resulting precipitate was filtered off, washed with cold water (50 ml.) and air dried. The yield of 7-amino-3-[S-(l,2,3- triazole-S-yl)thiomethyl]-3-cephem-4-carboxylic acid was 8 g., dec. pt. 230 C. IR analysis showed some decomposition of the B-lactam ring but it was used as is for the next step.

Anal. Calcd. for C H N O S C, 38.39; H, 3.54 Found: C, 38.36; H, 3.78.

Purification of 7-Amino-3-( l ,2,3-triazol-5-ylthiomethyl)-3-cephem-4- carboxylic acid (5h) Crude 7-amino-3-(1,2,3-triazol-5-ylthiomethyl)-3- cephem-4-carboxylic acid 16.1 g.) containing approximately 20 mole of 7-aminocephalosporanic acid as an imputity, was brought into solution with 600 ml. of methanol and 40 ml. of cone. HCl. After carbon treatment, the solution was diluted with 1.5 l. of ice water and extracted once with ethyl acetate. The aqueous phase was concentrated at reduced pressure to remove methanol. The cold aqueous concentrate was then adjusted slowly to pH 4.0 with 20% sodium hydroxide causing the product to crystallize. The product was collected by filtration, washed with water and methanol and dried in vacuo over phosphorus pentoxide; l 1.4 g. The NMR spectrum indicated that this product contained about 7 mole of 7-aminocephalosporanic acid as an impurity.

The above purification procedure was repeated on 1 1.4g. of the product using 425 ml. of methanol, 28 ml. of cone. HCl and 1 l. of ice water yielding 8.0 g. of product. The NMR spectrum was fully consistent for the desired product and indicated no trace of 7-aminocephalosporanic acid as an impurity.

Anal. Calcd. for C, ,H, N O S C, 38.42; H, 3.55; N, 22.40

Found: C, 39.06; 38.53; H, 3.56, 3.51; N, 22.05, 21.60; H O, 1.78.

7-Amino-3-[2-(1,3,4-thiadiazo1yl)-thiomethyl]-3- cephem-4-carboxy1ic acid (5i) COOH 2-Mercapto-1,3.4-thiadiazole The procedure of J. Goerdeler, .1. Ohm and O. Tegtmeyer, Berichte 89, 1534 (1956) was followed. To a solution of 160 ml. of 48% hydrobromic acid and 100 mg. of powdered copper at 7 was added slowly with alternation 13.6 g. (0.1 mole) of 2-amino-l,3,4- thiadiazole (Eastman) and 32 g. of sodium nitrite in small portions over a period of /2 hour. The mixture was stirred for 1 Va hours at and for 1 hour at room temperature. The mixture was then neutralized with 50% potassium hydroxide to pH 9.5. The mixture was filtered and the filtrate was continuously extracted with ether for 6 hours. The ether was evaporated to 15 mm (25) to a solid which was dissolved in 40 ml. of ethyl alcohol and treated with g. of thiourea. The solution was heated at reflux for 1 /2 hours. A solution of 4.5 g. of potassium hydroxide in 65 ml. of water was added and the mixture was heated at reflux for an additional 1 /2 hours. The alcohol was evaporated at mm. (32) and the aqueous residue was neutralized with concentrated hydrochloric acid to pH 3.5. After cooling for 2 hours in an ice bath 3.5 of 2-mercapto-l,3,4- thiadiazole as yellow crystals were collected and weighed 3.5 g. M.p. 125127. The IR and NMR spectra were consistent for the structure.

7-Amino-3-[2-( l ,3,4-thiadiazolyl)-thiomethyl]-3- cephem-4-carboxylic acid (5i) To a suspension of 8.1 g. (.03 mole) of 7-aminocephalosporanic acid and 3.5 g. (.03 mole) 2'mercapto- 1,3,4-thiadiazole in 200 ml. of .l M phosphate buffer (pH 6.5) was added with stirring 5.4 g. (.064 mole) of sodium bicarbonate. The mixture was stirred at under nitrogen and all of the solid dissolved. The stirring was continued for 3 hours and the solution was cooled to 5 and adjusted to pH 5 with glacial acetic acid. The mixture was stored for 2 hours and the product, 7-amino-3-[2-(1,3,4-thiadiazoly1)-thiomethyl]-3- cephem-4-carboxylic acid, was collected and weighed 9 g., M.p. 140 (slow decomp.). The IR and NMR spectra were consistent for the structure.

7-Amino-3-[2-(5-hydroxymethyl-1,3 ,4- thiadiazolyl)thiomethyl]3-cephem-4-carboxy1ic acid N cH,-s- CHZOH COOH 1Hydroxyacetylthiosemicarbazide Found: C, 24.08; H, 4.50; N, 27.67; 5, 21.35. The IR and NMR spectra were consistent for the structure.

2-Acetoxymethyl-5-amino-1,3,4-thiadiazole A mixture of 22.4 g. (0.15 mole) of l-hydroxyacetylthiosemicarbazide and ml. of acetyl chloride was stirred at room temperature for 2 hours and then heated to 40 until all the solid had dissolved (ca. 2 hours). The acetyl chloride was removed under reduced pressure 40 (15 min.) and the residue was stirred with ml. of ice water. The mixture was adjusted to pH 9.2 with 10% KOH and the product was collected. The crude solid was recrystallized from absolute alcohol to yield after air drying 6 g. of crystalline 2-acetoxymethyl-5-aminol ,3 ,4-thiadiazole, M.p. 191-192 C.

Anal. Calcd. for C H N O S: C, 34.63; H, 4.07; N, 24.26

Found: C, 35.16; H, 4.35; N, 24.38.

2-Hydroxymethyl-5mercapto-1,3 ,4-thiadiazole The procedure of Goerdler et al. was followed using 34.6 g. grams (0.2 mole) of 2-acetoxymethyl-5-amino- 1,3,4-thiadiazole and 64 grams (0.9 mole) of sodium nitrite in 320 ml. of 48% hydrobromic acid and 0.1 gram of powdered copper. The bromo compound was treated with 7.6 grams (0.1 mole) of thiourea and l 1.2 grams of potassium hydroxide in 25 ml. of water. Upon acidification with 6 N hydrochloric acid the mercaptan was extracted in ethyl acetate and treated with 17 grams of potassium 2-ethylhexanoate. The potassium salt was collected, washed with ethyl acetate and dried to weigh 12.2 grams. The salt was recrystallized from acetone-water to give 7.6 grams of crystalline potassium 2-hydroxymethyI-S-mercapto-1,3,4-thiadiazole, M.p. 130-131c.

Anal. Calcd. for C H KN S O.H O: C, 17.64; H, 2.46; N, 13.70

Found: C, 17.81; H, 2.43; N, 13.66.

The IR and NMR spectra were consistent for the structure.

7-Amino-3-[2-(5-hydroxymethyl-1,3,4- thiadiazolyl)thiomethyl]-3-cephern-4-carboxylic acid 1') The procedure followed was the same as the procedure for the unsubstitutedthiadiazole using 2.25 g. (0.012 mole) of mercaptan, 3.3 g. (0.012 mole) of 7-aminocephalosporanic acid and 1 g. (0.012 mole) of sodium bicarbonate in 100 ml., 1 M phosphate buffer to yield 3.15 g. of tan, solid-7-amino-3-[2-(5- hydroxymethyl-l ,3,4-thiadiazolyl )thiomethyl ]-3- cephem-4-carboxylic acid, M.p. 17017'5 C. decomp. The IR and NMR spectra were consistent for the structure.

Description of the Preferred Embodiments 7-[a-(2-t-Butoxycarbonylaminomethyl-1,4- cyclohexadienyl)acetamido]-3-(3-hydroxypyridazin-6 ylthiomethyl)-3-ccphem-4-carboxylic acid 6a) To a stirred solution of 1.1 g. (4.1 mmoles) of a-[2- (t-butoxycarbonylaminomethyl)-1 ,4-cyclohexadienyl acetic acid (3) and 800 mg. (4.4 mmoles) of 2,4- dinitrophenol in 40 ml. of ethyl acetate was added in one portion 0.9 g. (4.4 mmoles) of N,N'-dicyclohexy lcarbodiimide (DCC). The reaction mixture was stirred at room temperature for 3 hours. The separated dicyclohexylurea was filtered off. The filtrate was evaporated to dryness to give the oily activated ester which was dissolved in 30 ml. of THF. To the solution was added a solution of 1.26 g. (3.7 mmoles) of 7-amino-3- (3 -hydroxypyridazin-6-ylthiomethyl )-3 -cephem-4- carboxylic acid (5a) and 1.03 ml. (7.4 mmoles) of triethylamine in 20 ml. of 50% THF. The mixture was stirred at room temperature for 18 hours and concentrated in vacuo to 10 m1. of the volume. The concentrate was washed with five 100 ml. portions of ether, acidified with 6 N hydrochloric acid and extracted with six 100 ml. portions of ethyl acetate. The combined ex- 5 cc 3.03 21-1. s, CH2CO), 3.4 3.' I'(4H, m, 2-1-1 and Clil -N), 3.9 4.2 (2H, 3-Cl;l 498 11-1. d, 4.5 Hz, 6-H), 4.9 5.2 (3H, m, 5: 1E and c-Nn), 6.29

(1H, cl, 9.5 Hz, pyridazine-l-l), 7.23 (1H, d, 9.5 Hz,

pyridazine-H), 8.77 (1H, d, 7.5 Hz, CONlj).

Anal. CalCCl. for C26H3,N5O1S2.H2O:'C, 51.56; 11, 5.16; 11.56; s, 10.59

Found: c, 51.43; 11, 5.13; N, 11.66; 5, 10.79.

7-[a-(2-Aminomethyl-1,4,- cyclohexadienyl )acetamido]-3-( 3-hydroxypyridaz in-6- ylthiomethyl)-3-cephem-4-carboxylic acid (7a) A. A solution of 670 mg. (1.13 mmoles) of the BOC 'protected cephalosporin 6a in 1.5 ml. of trifluoro acetic acid was stirred at 0 C. for 1 hour. To the solution was added ml. of dry ether. The result- I ing precipitate was collected by filtration, suspended in 20 ml. of water and adjusted to pH 6 with dilute ammonium hydroxide to give a viscous oil, which was triturated with acetonitrile to give 380 mg. (75.5%) .of the desired product7a melting at 200-205 C. (dec.). i I 1R: 1765, 1640, 1580, 1390, 1350 cm. 7 Anal. Calcd.for C H N O S J/2H O: C, 50.59; H,

Found: C, 50.95; H, 4.79; N, 13.10 B. To a stirred suspension of 1.1 g. (3.6 mmoles) of sodium; 7 .{2-[N-( l-carbethoxypropen-Z- yl)aminomethyl]-l ,4-cyclohexadienyl acetate (4) in 20 ml. of dry tetrahydrofuran (THF) containing one drop of N,N-dimethylbenzylamine was added 0.38 ml. (3.95 mmoles) of ethyl chloroformate at l0 to 5 C. To the mixed anhydride solution was added in one portion a solution of 1.02 g. (3 mmoles) of 7- [a-(2 aminomethyl-1,4- cyclohexadienyl)-acetamido]-3-(3- hydroxypyridazin-6-ylthiomethyl)-3-cephem-4- carboxylic acid (5a) in 10 ml. of 50% aqueous THF containing 0.42 ml. (3 mmoles) of triethylamine. The mixture was stirred for 30 min. at room temperature, treated with active carbon and filtered. To the filtrate was added 3 ml. of formic acid and the mixture was stirred for30 min. at room temperature. The resulting precipitate was filtered, washed with water and acetonitrile to give 570 mg. of 7a EXAMPLE 2 b ]pyridazin-6-ylthiomethyl) 3-cephem-4-carboxylic To a stirred solution of 3.2 g. (0.012 mole) of a-[2- (t-butoxycarbonylaminomethyl)-1,4-cyclohexadienyl- ]acetic acid (3) and 0.3 g. (0.0125 mole) of 2,4- dinitrophenol in 50 ml. of THF was added 2.6 g. (0.0125 mole) of DCC and the mixture was stirred for 1.5 hours at room temperature. The precipitated urea was removed by filtration. A solution of 3.65 g. (0.01 mole) of 7-amino-3-(tetrazolo-[1,5-b]pyridazin-6- ylthiomethyl-3-cephem-4-carboxylic acid (b) and 2.8 ml. (0.02 moles) of triethylamine in 100 ml. of water was added to the active ester solution. The mixture was stirred for 18 hours at room temperature. Most of THF was removed under reduced pressure below 40 C. and the residual solution was washed with ether (3 X 50 ml.) and acidified with 6 N HCL and extracted with ethyl acetate (8 X 100 ml.). The combined extracts were washed with water (3 X 100 ml.) and saturated NaCl solution (2 X 100 ml.) and evaporated to dryness. The oily residue was triturated with ether to give 2.2 g. (52%) of 6b melting at 165 C. (dec.).

1R: v,,,,,,." 1770, 1710, 1670, 1510, 1240, 1150 cm Anal. Calcd. for C H N O S C, 50.18; H, 4.92; N, 18.23; S, 10.43.

Found: C, 51.05; H, 4.97; N, 17.24; S, 9.09.

7-[2-Aminomethyl-1,4-cyclohexadienyl)acetamido]-3- (tetrazolo[ 1,5-b]pyridazin-6-ylthiomethyl-3-cephem 4-carboxylic acid (7b) A mixture of 3.1 g. (5.05 moles) of 6b and 6 ml. of trifluoroacetic acid was stirred for one hour under cooling and diluted with 200 ml. of ether to separate the trifluoroacetate of 7b which was dissolved in 5 m1. of water, adjusted to pH 6 with conc. NH OH and diluted with 900 ml. of acetonitrile. The yellow precipitate was collected by filtration and washed with acetonitrile to give 2.3 g. (88.5%) of 7b melting at 210 C. (Dec.). Recrystallization of 2 g. of 7b from 350 ml. of 50% THF gave 1.19 g. of pale yellow needles melting at 220230 C. (dec.).

IR: v f 1785, 1640, 1610, 1570 cm".

Anal. Calcd. for C H N O S .1/2C H O: C, 50.17; N, 4.76; N, 20.35; S, 11.64.

Found: C, 50.17; H, 4.56; N, 19.59; S, 11.30.

EXAMPLE3 CHZNHZ H CHCO- 5 N-1Hl l 9 N com CH,

7-[(2-N-t-Butoxycarbonylaminomethyl-1,4- cyclohexadienyl)-acetamido]-3-( l-methyltetrazol-S- ylthiomethyl )-3-cephem-4-carboxylic acid (6c) To a stirred solution of 3.2 g. (0.012 mole) of a-[2 (t-butoxycarbonylaminomethyl)-1,4-cyclohexadienyl1- acetic acid (3) and 2.3 g. (0.012 mole) of 2,4- dinitrophenol in 50 ml. of THF was added in one portion 2.6 g. (0.012 mole) of DCC and the mixture was stirred for 1.5 hours at room temperature. The precipitated urea was removed by filtration. To the filtrate was added a solution of 3.28 g. (0.01 mole) of 7amino-3- 1-methyltetrazol-5-ylthiomethy1 )-3-cephem-4- carboxylic acid (5c) and 2.8 ml. (0.02 mole) of triethylamine in ml. of water and the mixture was stirred for 18 hours at room temperature. Most of THF was removed under reduced pressure below 40C. and the residual aqueous solution was washed with ether (2 X 50 ml.), acidified with 6 N HCl and extracted with ethyl acetate (4 X 50 ml.). The-combined extracts were washed with water (2 X 50 ml.) and a saturated NaCl solution (2 X 50 ml.), dried with Na SO and evaporated to dryness. The oily residue was triturated with ether (100 ml.) to give 2.3 g. (48%) of 6c, melting at C. (dec.).

1R: v,,,.,,."'' 1780, 1700, 1510, 1240, 1150 cm.

Anal. Calcd. for C H N O S C, 49,90; 1H, 5.41; N, 16.97; S, 11.10

Found: C, 50.23; H, 5.23; N, 15.80; S, 12.43

7-[ (2-Amin0methyll ,4-cyclohexadienyl )acetamido]- 3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4- carboxylic acid (7c) A mixture of 2.2 g. (3.8 mmoles) of 6c and 5 ml. of trifluoroacetic acid was stirred for one hour under cooling and diluted with 200 ml. of ether to separate the trifluoroacetate of 7c which was dissolved in 5 ml. of water. The solution was adjusted to pH 6 with conc. NH OH and diluted with 200 ml. of acetonitrile to give the yellow precipitate which was collected by filtration, washed with acetonitrile to yield 1.5 g. (82.5%) of 7c, melting at 200 C. (dec.), which was recrystallized from 50% THF to give pale yellow needles. M.p. 210 C. (dec.).

IR: v 1785, 1640, 1615, 1570 cm.

Anal. Calcd. for C H N O S J/2C H O: C, 49.11; H, 5.30; N, 19.09; S, 12.48

Found: C, 49.85; H, 5.20; N, 18.53; S, 12.26.

EXAMPLE4 CH2NH2 c i H s CH2CO-N N .N

N a swlcn, -O 1 coal 7-[(2-N-t-Butoxycarbonylaminomethyl-1,4- cyclohexadienyl)-acetamido]-3-(5-methyl-1,3,4- thiadiazol-2-ylthiomethy1)-3-cephem-4-carboxylic acid To a stirred solution of 900 mg. (3.3 mmoles) of a-[- 2-(t-butoxycarbonylaminomethyl)-1,4- cyclohexadienyl]-acetic acid (3) and 660 mg. (3.6 mmoles) of 2,4-dinitrophenol in 40 ml. of THF was added in one portion 740 mg. (3.6 mmoles) of DCC and the mixture was stirred for 2 hours at room temperature. The precipitated urea was removed by filtration. To the filtrate was added a solution of 1.03 g. (3 mmoles) of 7-amino-3-(5-methyl-1,3,4-thiadiazol-2- ylthiomethyl)-3-cephem-4-carboxylic acid (d) and 1.3 ml. (9 mmoles) of triethylamine in 30 ml. of water in one portion and the mixture was stirred for 18 hours at room temperature. The THF was removed under reduced pressure below 40 C. and the concentrate was washed with ether (3 X ml.) and acidified with 6 N HCL and extracted with ethyl acetate (5 X 10 ml.). The combined extracts were washed successively with water (2 X 10 ml.) and a saturated NaCl solution (10 ml.). Evaporation followed by trituration with ether gave 980 mg. (55%) of 6d.

IR: z/ 1770, 1710, 1660, 1610. 1510, 1240, 1150 cm Anal. Calcd. for C H N O S C, 50.57; H, 5.26; N, 1 1.80.

Found: C, 50.51; H, 5.20; N, 11.46.

7-[(2-Aminomethyl-1,4-cyclohexadienyl)acetamido]- 3-(5-methy1-1,3,4-thiadiazol-2-ylthiomethyl)-3- cephem-4-carboxylic acid (7d) A. A mixture of 940 mg. (1.58 mmoles) of 6d and 2 ml. of trifluoroacetic acid was stirred for one hour under cooling and diluted with 100 ml. of ether to separate the trifluoroacetate of 7d which was dissolved in 5 ml. of water and 5 ml. of acetonitrile. The solution was adjusted to pH 6 with conc. NH OH and diluted with 100 ml. of acetonitrile. The resulting pale yellow precipitate was collected by filtration and washed with acetonitrile (5 ml.) to give 600 mg. (77%) of 7d, melt-- ing at 230240 C. (dec.).

1R: v 1790, 1650, 1580 cm Anal. Calcd. for C H N O S C, 48.66; H, 4.70; N, 14.19; S, 19.49

. Found: C, 49.24; H, 4.59; N, 13.88; S, 20.16

B. To a stirred suspension of 1.1 g. (3.6 mmoles) of sodium {2-[N-( l-carbethoxypropen-Z- yl)aminomethyl]-1,4-cyclohexadieny1} acetate (4) in 20 ml. of dry THF containing one drop of N,N-dimethylbenzylamine was added 0.38 ml. (3.95 mmoles) of ethyl chloroformate for 10 min. at 10 to C. To the mixed anhydride solution was added in one portion a solution of 1.03 g. (3 mmoles) of 5d in 10 ml. of 50% aqueous THF containing 0.42 ml. (3 mmoles) of triethylamine and the mixture was stirred for 30 min. at room temperature. A few drops of formic acid was added and the mixture was filtered. To the filtrate was added 200 ml. of ether and 3 ml. of formic acid. The precipitate was collected by filtration, washed with water and acetonitrile to give 1.] g. (62%) of 7d.

7-[(2-Nt-Butoxycarbonylaminomethyl-1,4- cyclohexadienyl)acetamido]-3-(3- hydroxypyridazino[3,2-c]S-triazol6-ylthiomethy1)-3- cephem-4-carboxylic acid (6e) To a stirred mixture of 900 mg. (3.3 mmoles) of 01-1- 2-(t-butoxycarbonylaminomethyl)-1,4-cyclohexadienyl]acetic acid (3) and 660 mg. (3.6 mmoles) of 2,4-dinitrophenol in 30 ml. of THF was added in one portion 740 mg. (3.6 mmoles) of DCC and the mixture was stirred for 2 hours at room temperature. The separated urea was filtered off. To the activated ester solution was added in one portion a solution of 1.13 g. (3

mmoles) of 7-amino-3-(3-hydroxypyridazino[3,2-c]-striazol-6-ylthiomethyl)-3-cephem-4-carboxylic acid (5e) and 1.25 ml. (9 mmoles) of triethylamine in 30 ml. of water and the mixture was stirred for 18 hours at room temperature. The THF was distilled off under reduced pressure below 40 C. and the residue was washed with ether (2 X 10 ml.) and acidified with 6 N HCL and extracted with ethylacetate (5 X 10 ml.). The combined extracts were washed with water (2 X 10 ml.) and a saturated NaCl solution 10 ml. and evaporated to dryness. The residue was triturated with ether to give 505 mg. (27%) of 6e, melting at C. (dec.).

1R: v,,,,,,"'' 1770, 1700, 1510, 1200, 1150 cm".

Anal. Calcd. for C H N O S C, 51.50; H, 4.96; N, 15.57; S, 10.18

Found: C, 51.61; H, 4.80; N, 14.47; S, 10.25

7-[(2-Aminomethyl-1,4-cyclohexadienyl)acetamido]- 3-( 3-hydroxypyridazino-[3 ,2-cl-s-triazol-6- ylthiomethyl)-3-cephem-4-carboxylic acid (7e) Trifluoroacetic acid (1 ml.) was added to 470 mg. (0.747 mmoles) of 6e and the mixture was stirred for one hour under cooling and diluted with 100 ml. of ether to separate the trifluoroacetate of 7e which was dissolved in 5 m1. of water and adjusted to pH 6 with conc. NH OH. An addition of 100 ml. of acetonitrile to the mixture gave yellow precipitate which was collected by filtration and washed with acetonitrile to give 360 mg. (91%) of 7e, melting at 210220 C. (dec.).

1R: v,,.,,,"' 1760, 1710, 1640, 1580 cm".

Anal. Calcd. for C H N O S H O: C 48.25; H, 4.60; N, 17.91; S, 11.71

Found: C, 48.75; H, 4.60; N, 16.56; S, 12.17.

EXAMPLE 6 CH NH H s CHzCO-N? N N-N l O R N CO H 7-[(2-Nt-Butoxycarbonylaminomethyll ,4- cyclohexadienyl)-acetamido]-3-(pyrido[2,1-c]-striazol-3-ylthiomethyl)-3-cephem-4-carboxy1ic acid DCC (740 mg., 3.6 mmoles) was added to a solution of 950 mg. (3.3 mmoles) of a-[2-(tbutoxycarbonylaminomethyl)-1,4-cyclohexadienyl]acetic acid (3), and 660 mg. (3.6 mmoles) of 2,4- dinitrophenol in 30 ml. of THF and the mixture was was filtered off. To the filtrate was added a solution of 1.1 g. (3 mmoles) of 7-amino-3-(pyrido[2,1-c]-striazol-3-ylthiomethyl)-3-ylthiomethy1)-3-cephem-4- carboxylic acid (5f) and 1.25 ml. (9 mmoles) of triethylamine in 30 ml. of water and the mixture was stirred for 18 hours at room temperature. The THF was removed under reduced pressure below 40 C. and the residue was washed with ether (2 X ml.), acidified with 6 N HCl and extracted with ethyl acetate (5 X 10 ml.). The combined extracts were washed with water (2 X 10 m1.) and a saturated NaCl solution (10 ml.) and evaporated to dryness below 40 C. Trituration of the residue with ether gave 1.15 g. (63%) of 6f, melting at 5 cetic acid (3) 30 triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid g) To a solution of a-[ 2-( tbutoxycarbonylaminomethyl)-1,4-cyc1ohexadienyl]a- (1.07 g., 4 mmoles) and 2,4- dinitrophenol (0.74 g., 4 mmoles) in dry THF (10 ml.) was added DCC (0.82 g., 4 mmoles) and the mixture was stirred for 1 hour at room temperature at precipitate the urea which was removed by filtration. To the filtrate was added in one portion a cold solution of 7- amino-3-pyridazino[ 2,1 -c]-s-triazol-3-ylthiomethyl)-3- cephem-4-carboxylic acid (5g) (1.09 g., 3 mmoles) and triethylamine (0.81 g., 8 mmoles) in 50% aqueous THF (20 ml.) and the mixture was stirred for 20 hours at 145 150 C. (dec.). room temperature. The reaction mixture was washed IR: v 1770, 1680, 1510, 1240, 1 150 cm. with ether (2 X 50 ml.), acidified to pH 2 with diluted Anal. Calcd. for C H N O S C, 54.89; H, 5.26; N, hydrochloric acid and extracted with ethyl acetate (6 13.72; S, 10.47 X 50 ml.). The combined extracts were washed with Found: C, 53.84; H, 5.13; N, 13.12; S, 9.26. water (2 X 30 ml.), treated with carbon and dried on anhydrous sodium sulfate. Evaporation of the solvent gfigiz g z under reduced pressure afforded an oily residue which 4 carboxylic acid (7f) p was solidified by trituration with ether (50 ml.) to give 0.63 g. (34%) of 6g, M.p. 202213 C. (dec.).

Trifluoroacetic acid (2.5 ml.) was added to the BOC- IR mur 1785, 1780, 1520, 1250 1 160 Cmfl blocked cephalosporin 6f (1.1 g., 1.8 mmoles) with NMR; 8 ,139 (9H, s, CH -C), 3.89

EXAMPLE 7 I H s CH2C0N CHg-S O 7-[(2-N-t-Butoxycarbonylaminomethyl-1,4- cyclohexadienyl)-acetamido]-3-pyridazino[2,1 c]-s- H NH (ITHS cu coou cm-cl-o-c-rv (4H, s, H C 5.03 (1H, d, 4 Hz, 6-11), 5.67 (3H, br-s, 7-H and H C=), 7.20-8.90 (3H, m, pyridazin- H), 9.05 (1H, d, 8 Hz, NflCO).

Anal. Calcd. for C H ,N O S .H O: C, 48.08; H, 4.95; N, 17.83; S, 11.67

Found: C, 48.32; H, 3.94; N, 17.19; S, 11.90.

7-(2-Aminomethyl-1,4-cyclohexadienylacetamido)-3- (pyridazino[2,1-c]-s-triazol-3-ylthiomethy1)-3- cephem-4-carboxy1ic acid (7g) A mixture of 6g (0.53 g., 0.86 mmole) in trifluoroacetic acid (1 ml.) was stirred for 1 hour at 0 5 C. and diluted with ether (30 ml.) to precipitate the trifluoroacetate of 7g which was collected by filtration and dissolved in water (4 ml.). The solution was adjusted to pH 6 with ammonium hydroxide and dilute with acetonitrile ml.) to precipitate 7g which was collected by filtration, washed with acetonitrile (30 ml.) and dried in vacuo on P 0 Yield 0.38 g. (86%). 5 M.p. 208- 216 c. (dec.).

1R: v 1780, 1620, 1570, 1340 cm.

UV: k 276 nm (65600).

Anal. Calcd. for C H N O S .H O: C, 51.33; H, 5.27; N, 15.52; 5, 10.15

Found: C, 51.00; H, 5.10; N, 15.00; S, 10.12.

Reaction Scheme for Examples 8, 9 and 10 DCC NH CH NHIOC(CH3)3 N 1 A 60%- NH 5 'CH S-R l N o 0 H2SR COOH 0 cool-I CHZNHZ I Y 3+ cF.co.H- cH fi NH- 5 r coon 7 H S S -CH2OH N l I l I l 1 N I El L l h v EXAMPLE over P 0 at to yield 1.4 g. (65.4%) of white pow- 8 der. The 1R .and NMR spectra were consistent for the structure. M.p. 135 slow dec..

CHZNH2 7-[(2-Aminomethyl-l,4-cyclohexadienyl)acetamidol- 3-( 1,3 ,4-thiadiazole-2-mercaptomethyl)-3-cephem-4- S carboxylic acid (7i)' i v CHZCONH A solution of 1.30 g. (0.00226 mole) of (6i) and 3.0 N J ml. trifluoroacetic acid was stirred for 1 hr. at 0. The

solution was diluted with 200 ml. of ether and the pre- COOH S cipitate collected by filtration. The trifluoroacetate salt 40 was suspended in 40 ml. of water and adjusted to pH 6.0 with dilute ammonium hydroxide. The gummy residue was then triturated with 25 ml. of acetonitrile to give 370 mg. (34.5%) of white powder. The product was dried for 16 hr. in vacuo over P 0 at 25. The IR and NMR spectra were consistent for the structure.

7-[(2-tert.-Butoxycarbonylaminomethyl-1,4- cyclohexadienyl)acetamido]-3-( 1,3,4-thiadiazole-2- mercaptomethyl)-3-cephem-4-carboxylic acid (6i) M p 0 Slow dec. Anal:.calcd for CwH2lN5O4S3H2. To a stirred solution of 1.10 g. (0.0041 mole) of 3 C, 45.85; H, 4.65; N, 14.07

and 0.80 g. (0.0044 mole) of 2,4-dinitrophenol in 40 Found: C, 45.71; H, 4.52; N, 14.12-

ml. of ethyl acetate was added in one portion 0.90 g. Th ilibrium solubility of this compound in (0 44 m le) f N.N'-dicy y n mgrn./ml. was 4.28 in distilled water and 5.85 in pH 7.0 the mixture was stirred for 3 hr. at 25. The dicyclohexphosphate b ff r,

ylurea was collected and the filtrate was evaporated at 45 (15 mm) to give the activated ester as an-oil. The EXAMPLE 9 oil was dissolved in 30 ml. of THF. A solution of 1.22

g. (0.0037 mole) of 7-amino-3-(1,3,4-thiadiazole-2- CHzNH2 mercaptomethyl)-3-cephem-4-carboxy1ic acid and 1.03 ml. (0.0074 mole) of triethylamine in 20 ml. of

50% THF-water was added to the solution of the actis vated ester and stirred for 18 hr. at 25. The THF was CHZCOW removed at 40 15 mm.) and the concentrate (10 ml.) ,0 N CH2OH was washed with ether 5 X 100 m1.) and acidified to pH 2 with 40% phosphoric acid. The mixture was ex- COOH tracted with ethyl acetate (6 X 100 ml.) and the combined extracts were washed with water and finally with l a saturated sodium chloride solution. The ethyl acetate solution was evaporated at 40 (15 mm.) to a volume 7-[(2-Aminomethyl-l,4-cyclohexadienyl)acetamido]- of 20 ml. and diluted with 30 ml. of Skellysolve B. The 3-(5-hydroxymethyl-1,3,4-thiadiazole-2- product 6i was collected and dried for 16 hr. in vacuo mercaptomethyl)-3-cephem-4-carboxylic acid (7j) The procedure was the same as that of Example 8, except the product was collected from the water to obtain 340 mg. (29.8%) of a tan powder. A second fraction was obtained from the filtrate to give 230 mg. (20.0%) of 7j as a yellow crystalline hemi-hydrate. The IR and NMR spectra were consistent for the structure. M.p. 150 slow dec.

Anal. Calcd for C H N O S 1 1/2 H O: C, 44.75; H, 4.88; N, 13.05.

Found: C, 44.84; H, 4.63; N, 12.75.

The second fraction was found to be of equal purity. The [R and NMR spectra were consistent for the structure.

EXAMPLE l CH- NH- s CH2CO-NH (Hm-16: N cH, s c N 0 COOH l 7-[ (2-Aminomethyl-l ,4-cyclohexadienyl )acetamido]- 3-( ,2,3-triazole-5-mercaptomethyl )-3-cephem-4 carboxylic acid h The procedure was the same as that of Example 8, except the product was collected from ml. of acetonitrile to obtain 100 mg. (19.1%) of 7h as a light tan powder. The IR and NMR spectra were consistant for the structure. M.p. 160 slow dec.

Anal. Calcd. for C H N O S .2H O: C, 46.17; H, 4.88; N, 16.38

Found: C, 45.76; H, 5.25; N, 16.85.

EXAMPLE 1 1 Preparation of 7-[(2-Aminomethyl-1,4-cyclohexadieny1)-acetamido]- 3-( 1 -methyltetrazo1-5-ylthiomethyl )-3-cephem-4- carboxylic acid (70). Lot 2 EINH-BOC @NHBOC COOH COO@-NO2 NH-BOC El, H

CON NN N I ll CH S N 0 Dicyclohexylcarbodiimide (DCC) (9.0 g.. 0.044 mole) was added to a mixture of 1 1.2 g. (0.042 mole) of (2-N-t-butoxycarbonylamino- 1 ,4-cyclohexadieny1- )acetic acid (2) and 8.03 g. (0.044 mole) of 2.4- dinitrophenol in 250 ml. of THF. The mixture was stirred at room temperature for 1 .5 hr. and the precipitated urea was filtered off. To the filtrate was added a solution of 11.5 g. (0.035 mole) of 7-amino3-( 1- methyltetrazol-S-ylthiomethyl)-3-cephem-4-carboxy1ic acid and 14.7 ml. (ca. 0.105 mole) of triethylamine in 250 ml. of water. The mixture was stirred at room temperature for 20 hr. and the THF was removed under reduced pressure below 40 C. The aqueous solution was washed with ether (200 ml.), acidified with 6 N HCl and extracted with ethyl acetate (3 X 200 ml.). The extracts were washed with water and a saturated aqueous NaCl solution, dried with Na SO and evaporated to dryness. The oily residue was triturated with ether (500 ml.) to give 8.05 g. (40%) of the N-blocked (that is, 60).

IR: v,,,,,,.''"'' 1770, 1710, 1650, 1510, cm.

A mixture of 8.0 g. (0.0138 mole) of 6c and 16 ml. of trifluoroacetic acid (TFA) was stirred at 0-5 C. for 1 hour and then diluted with 300 ml. of ether to separate the trifluoroacetate of 7c, which was dissolved in 30 m1. of 50% acetonitrile, adjusted to pH 6 with cone. NH OH and diluted with 500 m1. of acetonitrile. The yellow precipitate which separated was washed well with acetonitrile (200 ml.) to give 5.2 g. (79%) amorphous 70 (lot 2). M.p. -205 C. (dec.).

IR: v,,,,,,'"'' 1780, 1650, 1620, 1560 cm.

UV: A 7 274 nm (e, 9100).

Anal. Calcd. for C H N O S H O: C, 46.05; H, 5.08; N, 19.78; S, 12.94

Found: C, 46.64; H, 4.60; N, 18.63; S, 12.59.

Solubility in 0.1 M phosphate buffer (pH 7): 9.4 mg./ml.

EXAMPLE 12 Preparation of 7-[(2-Aminomethy1-1,4-cyc1ohexadienyl)-acetamido1- 3-(5-methyl-1,3,4-thiadiazol-Z-ylthiomethyl)-3- cephem-4-carboxy1ic acid (7d), Lot 2 H s COW CO H H s CON NN N k i] CH2S H1 0 r co H s To a stirred solutionof 8.02 g. (0.03 mole) of 2-(N-tbutoxycarbonylaminomethyl)-1,4- cyclohexadienylacetic acid (2) and 5.6 g. (0.0304 mole) of 2,4-dinitrophenol in 80 ml. of dry THF was added 6.2 g. (0.0304 mole) of DCC at 5 10 C. in one portion and the mixture was stirred for 2 hr. at

room temperature. After removing the precipitated urea, the reaction mixture was mixed at C. with a solution of 9.4 g. (0.027 mole) of 7-amino-3-(5- methyl-1,3 ,4-thiadiazol-Z-ylthiomethyl )-3-cephem-4- carboxylic acid and 7.7 ml. (0.055 mole) of triethylamine in 30 ml. of water and 50 ml. of THF. The mixture was stirred for 20 hr. at room temperature and concentrated under reduced pressure to remove most of THF. The aqueous solution was treated with 2 g. of active carbon. The filtrate was covered with 100 ml. of ethyl acetate and acidified with HCl. The aqueous layer was extracted with two 50 ml. portions of ethyl acetate. The ethyl acetate layer was combined with the ethyl acetate extracts, washed with two 100 ml. portions of water, dried with anhydrous Na SO and concentrated to dryness below 40 C. to give 6d as a viscous oil which solidified by trituration with ether to give 6d. Yield 12.5 g. (70%). IR: v 1780, 1700, 1680, 1510 cm.

The BOC-protected cephalosporin 6d 12.5 g., 0.021 mole) was treated with 25 ml. of TFA at 0 5 C. with stirring. Stirring was continued for one hour at the same temperature. The mixture was diluted with 300 ml. of ether to give white precipitate which was dissolved in 30 ml. of 50% aqueous acetonitrile and treated with 1 g. of active carbon. The filtrate was ad-' EXAMPLE 13 Preparation of 7-[(2-aminomethyl-l ,4-cyclohexadienyl)-acetamido]- 3-( 3-hydroxypyridazino- 3 ,2-c -s-triazol-6- ylthiomethyl)-3-cephem-4-carboxylic acid (7e), Lot 2 @zmpsoc @gNH-BOC COOH coop. N02

NHBOC (I H s CON N cu s N o COOH NH, oc H 5 CON N l CH- .S N o coon To a stirred solution of 10.1 g. (0.038 mole) of a-[Z- (t-butoxycarbonylaminomethyl )-l ,4-cyclohexadienyl]- acetic acid (2) and 8.1 g. (0.044 mole) of 2,4- dinitrophenol in 300 ml. of THF was added 9.1 g. (0.044 mole) of DCC in one portion and the mixture was stirred for 2 hr. at room temperature. The separated urea was filtered off. To the filtrate was added a solution of 14.4 g. (0.038 mole) of 7-amino-3-(3- hydroxypyridazino-[ 3 ,2-c]-s-triazol-6-ylthiomethyl )-3- cephem-4-carboxylic acid and 12.2 g. (0.12 mole) of triethylamine in 250 ml. of water and the mixture was stirred for 16 hr. at room temperature. The reaction mixture was evaporated under reduced pressure below 40 C. and the aqueous concentrate was washed with ether (5 X 200 ml.) and treated with active carbon. The

filtrate was acidified with dil. HCl to pH 2 to give a precipitate which was washed with 200 ml. of water and extracted with hot THF (5 X 500 ml'.). The THF extracts were evaporated under reduced pressure below 40 C. and the residue was triturated with 500 ml. of ether to give the N-blocked 7e (that is, 6e). IR: v,,,,,,." 1765, 1700, 1340, 1240, 1150 cm.

To the BOC-blocked cephalosporin 6e (7.8 g., 0.0124 mole) was added dropwise 16 ml. of TFA at 5 C. and the mixture was stirred for 1.2 hr. at room temperature. The mixture was diluted with 1000 ml. of ether and the resulting precipitate was dissolved in 50 ml. of 50% acetonitrile. The solution was adjusted at pH 4 5 with cone. NH OH and diluted with 200 ml. of acetonitrile to give 5.55 g. (84%) of 7e (Lot 2). M.p. 220 230 C. (dec.).

1R: v,,,,,,' 1760, 1710, 1640, 1540, 1460 cm.

UV: A J 7 253 nm (e, 20,000)

Anal. Calcd. for C H N-,O S .22H O: C, 47.06; H, 5.03; N, 17.46 Found: C, 46.28; 46.37; H, 3.73; 3.71; N,

Solubility in 0.1 M phosphate buffer (pH 7): 1.1 mg./ml.

Reprecipiration Trifluoroacetic acid (0.5 ml.) was added to 7e (lot 2, mg.) at 5 C. and the mixture was stirred for 5 min. at room temperature. The solution was diluted with 50 ml. of ether. The resulting trifluoroacetate was dissolved in 10 m1. of 50% aqueous acetonitrile solution. The solution was adjusted at pH 6.0 with 10% NH OH to give a light brown precipitate (91 mg.), m.p. 224234 C. (dec.).

1R: 1/,,,,,,"' 1760, 1710, 1630, 1540, 1460 cm" UV: h z a 7 253 nm (e, 19,000).

37 EXAMPLE 14 The salicylaldehyde adduct of 7-[ 2-aminomethyl-l ,4-cyclohexadienyl )acetamido 3-(1methyltetrazol-Sylthiomethyl)-3-cephem-4- .carboxylic acid (70) CO K CH3 To a stirred suspension of 7c (Example 1 1, lot 2, 766 mg., 1.6 mmoles) and triethylamine (300 mg., 3 mmoles) in methanol (8 ml.) was added salicylaldehyde (370 mg., 3 mmoles) and the mixture was stirred for 30 min. at room temperature to make a clear dark yellow solution. The solution was treated with a small amount of carbon and KEH (2 ml., 1 M solution in ethyl acetate of potassium 2-ethylhexanoate) was added to the filtrate. The mixture was diluted with a large amount of ether 100 ml.) to precipitate the product 80 which was collected by filtration, washed with ether (30 ml.) and dried. Yield 788 mg. (78%). M.p. 165-175 C. (dec.).

1R: v 1760, 1660, 1625, 1605, 1280 cm.

UV: e 7 256 nm (e 19500), 275 nm 14000, sh), 325 nm (e 2600).

NMR: 8,, 3.95 (3H, s, N-CH 4.96 (1H, d, 4 Hz, 6-H), 5.50 (1H, m, 7H), 5.69 (2H, s, C=C-H), 6.7 7.6 (4H, m, phenyl-H), 8.68 (1H, s, -CH=N-), 9.00 (1H, d, 8 Hz, 7-CONH).

Anal. Calcd. for C H O N S KH O: C, 48.96; H, 4.43; N, 15.37; S, 70.06.

Found: C, 48.96; 48.85; H, 4.01, 4.10; N, 14.33, 14.26; S, 9.31, 9.70.

EXAMPLE 15 The salicylaldehyde adduct of 7-[ 2-aminomethyl-l ,4-cyclohexadieny1)acetamidol- 3-( S-methyl- 1 ,3,4-thiadiazol-Z-ylthiomethyl)-3- cephem-4-carboxylic acid (7d) N=CH E-- CONH- s N CH -Sk s O COZH S To a stirred mixture of 7d (Example 12, lot 2, 778 mg., 1.6 mmoles) and triethylamine (300 mg., 3 mmoles) in methanol (8 ml.) was added salicylaldehyde (370 mg., 3 mmoles) and the suspension was stirred for 15 min. at room temperature to make a clear dark yellow solution which was treated with a small amount of carbon. KEH (2 ml., 1 M solution in ethyl acetate) was added to the filtrate. The mixture was diluted with a large amount of ether 100 ml.) to precipitate the product 8d which was collected by filtration, washed with ether (30 ml.) and dried. Yield 855 mg. (84%). M.p. 163-170 C. (dec.).

IR: v 1765, 1630, 1610, 1280 cm.

UV: A 7 257 nm (6 19800), 275 nm (e 14200, sh), 325 nm (e 2700).

10 g 6.76-7.60 (4H, m, phenyl=H 8.65 (1H, s, c 1-1= N), 8.92 (H, d, 8 Hz, CON Ii).

Anal. Calcd. for C H O N S K3/2H O: C, 48.92;

H, 4.41; N, 10.57; S. 14.51

Found: C. 48.91, 49.24; H, 3.74, 4.04; N, 10.15, 10.34; S, 12.75, 13.57.

EXAMPLE 16 The salicylaldehyde adduct of 7-[(2-aminomethyl-l ,4-cyclohexadienyl )acetamidol- 3-(3-hydroxypyridazino-[3,2-c]-s-triazol-6- ylthiomethyl)-3-cephem-4-carboxylic acid (7e) CO K 'To a stirred suspension of 7e (Example 13, lot 2, 754 mg., 1.42 mmoles) and triethylamine (300 mg., 3 mmoles) in N,N-dimethylformamide (7 ml.) was added salicylaldehyde (370 mg., 3 mmoles) and the suspension was stirred for 1.5 hr. at room temperature to be a clear dark yellow solution which was treated with a small amount of carbon. KEH (1.5 ml., 1 M solution in ethyl acetate) was added to the filtrate. The mixture was diluted with a large amount of ether (100 ml.) to precipitate the product 8e which was washed with ether (30 ml.) and dried. Yield 906 mg. M.p. 215-222 C. (dec.). 1R: v,,,,,,"' 1760, 1720, 1660, 1615, 1600, 1345, 1275 cm.

UV: h 7 255 nm (e 28800) 310 nm (e 8500, sh).

NMR: S s 5.00 (1H, d, 4 Hz, 6-H), 5.50 (1H, m, 7-l;1), 5.72 (2H, s, C=C-H), 6.7 7.6 (6H, m, phenyl-H), 8.70 (1H, s, CH=N), 9.00 (1H, d, 8H2, CONH).

Anal. Calcd. for C H O N S KH O: C, 50.49; H, 4.09; N, 14.21; S, 9.30

Found: C, 50.34, 50.30; H, 4.15, 4.00; N, 14.02, 14.03; S, 9.36, 9.31.

EXAMPLE 17 The potassium salt of the compound of Example 1 was prepared by adding a solution of potassium 2- ethylhexanoate (KEH) in ethyl acetate to a solution of the cephalosporin (zwitterion) (7a) in DMSO to precipitate the desired potassium salt.

EXAMPLE 18 The sodium salt of the compound of Example 3 was prepared by pre-formation of its diethylammonium salt in methanol followed by addition of a solution of sodium Z-ethylhexanoate (SEH) in ethyl acetate and then dilution with isopropanol to precipitate the desired so- 

1. IN ACID HAVING THE FORMULA
 2. The acid of claim 1 having the formula
 3. The sodium salt of the acid of claim
 2. 4. The potassium salt of the acid of claim
 2. 5. The zwitterion form of the acid of claim
 2. 6. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 2. 7. The acid of claim 1 having the formula
 8. The sodium salt of the acid of claim
 7. 9. The potassium salt of the acid of claim
 7. 10. The zwitterion form of the acid of claim
 7. 11. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 7. 12. The acid of claim 1 having the formula
 13. The sodium salt of the acid of claim
 12. 14. The potassium salt of the acid of claim
 12. 15. The zwitterion form of the acid of claim
 12. 16. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 12. 17. The acid of claim 1 having the formula
 18. The sodium salt of the acid of claim
 17. 19. The potassium salt of the acid of claim
 17. 20. The zwitterion form of the acid of claim
 17. 21. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 17. 22. The acid of claim 1 having the formula
 23. The sodium salt of the acid of claim
 22. 24. The potassium salt of the acid of claim
 22. 25. The zwitterion form of the acid of claim
 22. 26. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 22. 27. The acid of claim 1 having the formula
 28. The sodium salt of the acid of claim
 27. 29. The potassium salt of the acid of claim
 27. 30. The zwitterion form of the acid of claim
 27. 31. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 27. 32. The acid of claim 1 having the formula
 33. The sodium salt of the acid of claim
 32. 34. The potassium salt of the acid of claim
 32. 35. The zwitterion form of the acid of claim
 32. 36. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 32. 37. The acid of claim 1 having the formula
 38. The sodium salt of the acid of claim
 37. 39. The potassium salt of the acid of claim
 37. 40. The zwitterion form of the acid of claim
 37. 41. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 37. 42. The acid of claim 1 having the formula
 43. The sodium salt of the acid of claim
 42. 44. The potassium salt of the acid of claim
 42. 45. The zwitterion form of the acid of claim
 42. 46. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 42. 47. The acid of claim 1 having the formula
 48. The sodium salt of the acid of claim
 47. 49. The potassium salt of the acid of claim
 47. 50. The zwitterion form of the acid of claim
 47. 51. A nontoxic, pharmaceutically acceptable salt of the acid of claim
 47. 